The protein p53 is an important tumor suppressor. Under normal conditions, cells do not contain high levels of this protein. If a healthy cell is damaged, p53 is expressed and its cellular level increases, followed by inhibition of cell growth or programmed cell death (apoptosis). In order to induce apoptosis, p53 must bind to a specific DNA sequence. Numerous studies have demonstrated that p53 plays a very important role in directing a cell to stop growing or to undergo apoptosis. Therefore, p53 has been recognized as one of the most important guardians in the body to prevent damaged cells from developing into tumors. In approximately 50% of human tumors, a mutated form of p53 (mutant p53) is present that is unable to bind target DNA sequences, allowing an unregulated growth and division of such tumor cells. Indeed, mutation of p53 is considered the most frequent genetic alteration occurring in human cancer. Further, tumors associated with mutant p53 are often more resistant to chemotherapy than tumors with wild-type p53.
However, once a mutant p53 protein regains the ability to bind DNA, the tumor suppressor activity is restored and apoptosis is induced, consequently killing the cancer cell. The potential for small organic molecules to reactivate mutant forms of this protein has created a revolutionary new strategy for attacking cancer. In tumor cells, mutant forms of p53 are commonly present at elevated levels compared to wild-type p53 in healthy cells. This imbalance offers the possibility of selectively killing cancer cells over healthy cells via reactivation of mutant p53, which could lead to medications without the devastating side effects often associated with conventional chemotherapy. In addition to potential clinical applications, the ability to reactivate mutant p53 has also generated a fundamentally new approach in biomedical research: the possibility of designing organic molecules to re-establish the normal functions of a mutated protein.
Random screenings of combinatorial libraries have identified a handful of small organic molecules that affect the activity of p53 (FIG. 1). One of these molecules (PFTA) inhibits the DNA binding activity of wild-type p53, while others (e.g., CP-31398, CP-257042, PRIMA-1) restore or reactivate the DNA-binding activity of mutant p53 proteins. Although each of these molecules is structurally very different, there are common features among them that indicate the types of molecules to target p53. For example, each of these molecules has one or more amine functional groups that convey a net cationic charge under aqueous or physiological conditions. Most of these compounds also have aromatic groups, suggesting that aromatic-aromatic interactions promote binding to p53. In compounds CP-31398 and CP-257042, aromatic portions of these molecules are similar to known compounds that intercalate between the bases of DNA (such as ethidium bromide and acridine). It has been suggested that these molecules restore DNA-binding activity to p53 by binding simultaneously to both DNA and p53. However, while intercalation of aromatic groups into DNA is a favorable process, aromatic intercalators can also have mutagenic properties and thus are not suitable motifs for further development.
Among the compounds in FIG. 1, PRIMA-1 is an interesting target: it lacks an aromatic group (and therefore should not intercalate into DNA), yet it still restores DNA-binding activity to mutant p53. Although detailed knowledge about the interactions of PRIMA-1 with p53 are not known, it most likely involves a positioning of cationic charge and hydrogen bonding groups into proper orientations for p53 protein binding. This binding event may somehow induce a conformational change in mutant p53 such that DNA binding is restored. Alternatively, PRIMA-1 could also interact with a different protein target that then affects p53. However, the chemistry en route to PRIMA-1 precludes most structural analogs. The synthesis of PRIMA-1 does not readily allow introduction of chemical modifications, limiting access to analogs that target different mutant forms of p53 or derivatives that probe the mechanism of action.
P53 reactivation has been an on-going concern in the art. As outlined above, new approaches in organic chemistry are needed to synthesize new molecules that interact with p53. By examining the activity of such novel compounds, structure-activity relationships can be determined that will provide crucial information for development of new medications and understanding the mechanism(s) of action. These results can provide important insights into p53 reactivation and possibly new cancer chemotherapies.